Immunoglobulin (Ig) and T cell receptor (TCR) variable region genes are assembled from component V, D, and J gene segments in developing T and B lineage cells by the V(D)J recombination reaction. V(D)J recombination is tightly regulated to effect lineage - and developmental stage-specific, as well as allelically-excluded, patterns of Ig and TCR gene assembly via a mechanism based on modulating accessibility of substrate V, D, and J segments from different loci to a common V(D)J recombinase. Accessibility control is a broadly relevant process in developmental biology; and control of V(D)J recombination has provided one of the most useful paradigms. Yet, specific mechanisms that underlie accessibility control of Ig, TCR and other loci remain elusive. In particular, there is still little understanding of how cis elements within endogenous Ig or TCR loci work to regulate accessibility of specific V. D, and J segments over large chromosomal regions. This application proposes to approach this problem through the use of methods that, in large part, involve introduction of endogenous TCR and Ig locus mutations into ES cells followed by assays for the effects of these mutations in normal lymphocytes via a novel blastocyst complementation method. A major goal will be to elucidate novel mechanisms by which the recombination signal sequences (RSSs) that flank all V, D, and J segments direct the rearrangement of particular types of gene segments in a manner much more specific than previously appreciated. Additional goals will be to tailor (simplify) the large-scale structure of the endogenous TCRB and IgH loci to forms more amenable to analysis. These tailored loci will then be used as substrates for mutational and other types of analyses to systematically elucidate the elements and pathways that affect the locus-specific rearrangement patterns observed in normal development. With the accumulation of new information and technologies, we argue that transgenic/targeted mutation approaches can be refined to allow generation of significant new insights into processes that control antigen receptor gene assembly. Besides fundamental relevance to developmental biology, elucidation of V(D)J recombination control mechanisms has substantial implications for health and disease. Thus, these mechanisms clearly play a role in the development of normal antibody and TCR repertoires. Correspondingly, defects in V(D)J recombination may well underlie various immunological diseases ranging from immunodeficiencies to auto-immunity. Finally, aberrant V(D)J recombination has the potential to unleash oncogenic activities via translocations.